Carole Lemarié

Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

ABSTRACT There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.

The virulence variability of different Acinetobacter baumannii strains in experimental pneumonia.

OBJECTIVES Our objective was to compare the virulence of 5 strains of Acinetobacter baumannii by using a mouse model of pneumonia. METHODS Six-week old female C3H/HeN mice were used. The pneumonia was inducted by intra-tracheal inoculation of 5.10(6) bacteria. Spontaneous outcome was evaluated by mortality, mice weight variations, and a clinical score. Bacterial counts in lungs, spleen and blood, and inflammatory response in lungs (dosages of tumor necrosis factor-alpha and macrophage inflammatory protein-2) were also measured. Lastly, a histological examination of lungs was performed for 3 strains, giving a histological score. RESULTS Global mortality varied from 13% to 79% (P<10(-4)). Bacterial counts in lungs within the 4 days following inoculation varied significantly according to different strains. The evolution curves of bacterial counts were also different. There was a significant correlation between the clinical score and mortality (P<0.05) but not between bacterial counts in lungs and mortality. The increase of pro-inflammatory mediator production in lungs and the histological score also varied according to strains. CONCLUSIONS These results demonstrate the variability of the virulence between strains, and suggest that bacterial proliferation is not the only virulence factor responsible for the pathogenesis in A. baumannii pneumonia.

First report of blaNDM-1-producing Acinetobacter baumannii isolated in Lebanon from civilians wounded during the Syrian war

OBJECTIVES The emergence of carbapenem-resistant Acinetobacter baumannii has been observed worldwide. We describe the first detection of A. baumannii carrying the blaNDM-1 gene in Lebanon, isolated from Syrian patients wounded during the civil war. METHODS Four carbapenem-resistant A. baumannii strains isolated in 2012 in the Tripoli Government Hospital, Lebanon, from civilians wounded during the Syrian war, were analysed. Susceptibility was determined by disk diffusion testing, and resistance to carbapenems was confirmed by Etest. The presence of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, and blaNDM was investigated by PCR. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and blaOXA-51 sequence-based typing. RESULTS All isolates harboured the blaNDM-1 gene and were negative for other tested carbapenemases. They all belonged to the sequence type 85 and formed a single cluster by PFGE. Finally, blaOXA-51-like gene sequencing revealed the presence of the blaOXA-94 variant in all four isolates. CONCLUSION These findings show that Syria constitutes a reservoir for NDM-1-producing bacteria. These results also highlight the need for effective measures to stop the threatening spread of such strains.

How Many Samples and How Many Culture Media To Diagnose a Prosthetic Joint Infection: a Clinical and Microbiological Prospective Multicenter Study

ABSTRACT Although numerous perioperative samples and culture media are required to diagnose prosthetic joint infection (PJI), their exact number and types have not yet been definitely determined with a high level of proof. We conducted a prospective multicenter study to determine the minimal number of samples and culture media required for accurate diagnosis of PJI. Over a 2-year period, consecutive patients with clinical signs suggesting PJI were included, with five perioperative samples per patient. The bacteriological and PJI diagnosis criteria were assessed using a random selection of two, three, or four samples and compared with those obtained using the recommended five samples (references guidelines). The results obtained with two or three culture media were then compared with those obtained with five culture media for both criteria. The times-to-positivity of the different culture media were calculated. PJI was confirmed in 215/264 suspected cases, with a bacteriological criterion in 192 (89%). The PJI was monomicrobial (85%) or polymicrobial (15%). Percentages of agreement of 98.1% and 99.7%, respectively, for the bacteriological criterion and confirmed PJI diagnosis were obtained when four perioperative samples were considered. The highest percentages of agreement were obtained with the association of three culture media, a blood culture bottle, a chocolate agar plate, and Schaedler broth, incubated for 5, 7, and 14 days, respectively. This new procedure leads to significant cost saving. Our prospective multicenter study showed that four samples seeded on three culture media are sufficient for diagnosing PJI.

Wide spread of OXA-23-producing carbapenem-resistant Acinetobacter baumannii belonging to clonal complex II in different hospitals in Lebanon

OBJECTIVES To investigate the molecular epidemiology of Acinetobacter baumannii strains isolated from different hospitals in Lebanon. METHODS A total of 119 non-duplicate Acinetobacter strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and partial rpoB gene sequencing. Antibiotic susceptibility testing was performed by disc diffusion method and all identified carbapenem-resistant isolates were investigated by PCR assays for the presence of the carbapenemase-encoding genes. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for molecular typing. RESULTS Of the 119 A. baumannii isolates, 76.5% were resistant to carbapenems. The most common carbapenemase was the OXA-23-type, found in 82 isolates. The study of population structure using MLST revealed the presence of 30 sequence types (STs) including 18 new ones, with ST2 being the most commonly detected, accounting for 61% of the isolates typed. PFGE performed on all strains of ST2 identified a major cluster of 53 isolates, in addition to three other minor clusters and ten unique profiles. CONCLUSIONS This study highlights the wide dissemination of highly related OXA-23-producing carbapenem-resistant A. baumannii belonging to the international clone II in Lebanon. Thus, appropriate infection control measures are recommended in order to control the geographical spread of this clone in this country.

Assessment of the usefulness of performing bacterial identification and antimicrobial susceptibility testing 24 h a day in a clinical microbiology laboratory

The impact of inoculating agar media with positive blood cultures and of performing bacterial identification and antimicrobial susceptibility testing (AST) for positive urine cultures, blood cultures and certain fluid cultures after day hours (night service (NS)) was evaluated in a clinical microbiology laboratory. The impact of the NS was assessed in terms of decreases in the delays from the time of sampling to the time at which results became available and of the consequences for patient management and antimicrobial treatment. Two major benefits were obtained: initiation of earlier appropriate treatment, and change to a reduced-spectrum but still efficient regimen. The hours of laboratory testing and the availability and transmission of results to the clinical staff were recorded. Concurrently, these hours were estimated as though laboratory tests had been performed in the absence of NS. Reductions in delay were defined as the differences between the hours actually spent and the estimated hours. Economic concerns were also considered. Overall, 430 samples for which an identification and/or AST were performed during the NS were included in the study. The NS led to the implementation of earlier appropriate therapy in 97 cases (22.6%), and to the change to reduced-spectrum but still efficient regimens in 23 additional cases (5.3%). In conclusion, there appeared to be benefits from a system providing bacterial identification and AST overnight, but a study of the cost-effectiveness of the NS would be useful to back up this observation.

First report of carbapenemase-producing Acinetobacter baumannii carriage in pets from the community in France

Acinetobacter baumannii is responsible for nosocomial infections but can also cause community-acquired infections. Communityacquired infections are more prevalent in intertropical areas [1] and have been reported in survivors of natural disasters such as earthquakes or tsunamis as well as in soldiers and civilians duringwarfare. These latter observations support the assumption of an extrahospital reservoir of A. baumannii, but its epidemiology outside the hospital remains unclear. One potential community reservoir of A. baumannii is animals. A. baumannii is known as an agent of nosocomial infection in veterinarymedicine. By contrast, little is known regarding A. baumannii carriage incompanionanimals in thecommunity. Twopreviousstudies showed a high prevalence of A. baumannii carriage in companion animals of La Reunion Island (France) [2,3]. These studies were conducted in a tropical area, known as a potential predisposing factor for A. baumannii infection and carriage [1]. Conversely, few data are available for A. baumannii carriage in companion animals in temperate climates. The objective of this studywas to assessA. baumannii carriage in domesticated animals coming for a consultation in the veterinarian school of Nantes (ONIRIS), in metropolitan France. From April 2015 to June 2015, 150 pets (46 cats and 104 dogs) that consulted in the preventive medicine service were included in the study andwere sampled bymouth and rectal swabbing. A questionnaire about the animalwas also completedby theowner. Cultures were performed and A. baumannii colonies were identified using a VITEK®MS Plus Identification System (bioMérieux, Marcy-l’Étoile, France) and rpoB gene sequencing. Of the 150 animals, 4 carried A. baumannii (2.7%; 95% confidence interval 0.1–5.3%). These animals were dogs living in Nantes city or its surrounding areas (Table 1). Strains isolated from Dogs 1 and 2 were resistant to penicillins and penicillin/β-lactamase inhibitor combinations but remained susceptible to ceftazidime according to the Antibiogram Committee of the French Society for Microbiology (CASFM) recommendations (http://www.sfm-microbiologie.org/UserFiles/files/casfm/CASFMV2 _030915.pdf). They were also resistant to aminoglycosides, trimethoprim/sulfamethoxazole, ciprofloxacin and doxycycline. Both strains were highly resistant to imipenem [minimum inhibitory concentration (MIC) > 32 μg/mL], doripenem (MIC > 32 μg/mL) and meropenem (MIC > 32 μg/mL) as determined by Etest (bioMérieux). These strains were positive for the blaOXA-23 gene and were negative for blaOXA-24, blaOXA-58 and blaNDM by real-time PCR. Conversely, strains isolated from Dogs 3 and 4 were susceptible to the majority of antibiotics tested. Among the four A. baumannii strains, the Institut Pasteur multilocus sequence typing (MLST) scheme showed that the two carbapenem-resistant strains belonged to ST25; another strain belonged to ST250 and the last strain to a new sequence type, ST753. Pulsed-field gel electrophoresis (PFGE) analysis using the restriction enzyme ApaI revealed that the two ST25 A. baumannii strains had 100% homology, conclusive of a unique clone. The rate of A. baumannii carriage (2.7%) was lower than in La Reunion (6.5% [2] and 8.5% [3]). This difference may be partially explained by climate influence. Indeed, community-acquired A. baumannii infections are mainly reported in tropical climates [1]. La Reunion Island is located in a tropical climate, whilst the current study was conducted in metropolitan France, a temperate climate. Chen et al studied the impact of climatic variations on communityacquired A. baumannii pneumonia in humans and demonstrated that the prevalence of A. baumannii infections was higher during warm and humid months of the year [1]. We can suggest here that climate could also have an impact on A. baumannii carriage in companion animals. Further studies are necessary to assess this conclusion. Here we describe the first case of carbapenem-resistant A. baumannii carriage in non-hospitalised companion animals. The blaOXA-23 gene reported here has also been described in A. baumannii in animals, such as in hospitalised horses [4]. An OXA-23-producing A. baumannii strain has also been described in a cat hospitalised for a urinary tract infection [5]. This cat was infected and received antibiotherapy the month preceding the swabbing. Conversely, the pets included in the current study had no history of previous infections or antibiotic treatments. This observation raises questions about the extent of the circulation of carbapenemase-producing A. baumannii in companion animals in metropolitan France. The two carbapenem-resistant strains were similar by PFGE analysis. We do not exclude a potential cross-contamination of the two dogs. The two dogs, sampled the same day, could have been sampled in the same consultation box, hypothetically contaminated by this A. baumannii clone. No samplingwas performed in the boxes to verify this hypothesis. However, considering the practices of the professionals involved in the pet outpatient clinic, the risk of crosstransmission seems particularly weak. In conclusion, to our knowledge we describe here the first report of OXA-23-producing A. baumannii carriage in domesticated animals in metropolitan France. Further investigations must be conducted to assess A. baumannii carriage in pets in metropolitan France and to identify transmission mechanisms within the animals and also with their owners and their environment.

Diversity of Acinetobacter baumannii strains isolated in humans, companion animals, and the environment in Reunion Island: an exploratory study

OBJECTIVES Acinetobacter baumannii can be responsible for community-acquired infections in tropical climates like that of Reunion Island. The epidemiology of these community-acquired A. baumannii infections is not well understood. The aim of this study was to characterize A. baumannii strains isolated from patients at the time of admission to the university hospital of Saint-Denis, from environmental samples, and from pets. METHODS In this exploratory study, samples were collected by swabbing the rectum and mouth. A. baumannii isolates from positive samples were identified by VITEK 2 system, blaOXA-51-like gene PCR, and partial sequencing of the rpoB gene. Antimicrobial susceptibility testing was then performed. Strains were further analysed by multilocus sequence typing and pulsed-field gel electrophoresis. RESULTS A high prevalence of A. baumannii carriage was found in pets (8.5%). Only one A. baumannii isolate was resistant to carbapenems (isolated from a patient). A wide variety of A. baumannii, assigned to different sequence types, were isolated from pets, humans, and the environment. CONCLUSIONS This study shows that A. baumannii strains are present outside the hospital setting in Reunion Island and show great diversity. Further studies are needed to explore these extra-hospital reservoirs of A. baumannii in Reunion Island in greater detail and to determine their possible means of dissemination.

Association between an index of consumption of hand-rub solution and the incidence of acquired meticillin-resistant Staphylococcus aureus in an intensive care unit

Auteur Eveillard, Matthieu [1], Kouatchet, Achille [2], Rigaud, A. [3], Urban, M. [4], Lemarié, Carole [5], Kowalczyk, J.-P. [6], Mercat, Alain [7], Joly-Guillou, MarieLaure [8] Editeur WB Saunders Type Article scientifique dans une revue à comité de lecture Année 2009 Langue Anglais Date 2009/03 Numéro 3 Pagination 283 285 Volume 71 Titre de la revue Journal of Hospital Infection ISSN 0195-6701 URL de la notice http://okina.univ-angers.fr/publications/ua3521 [9] DOI 10.1016/j.jhin.2008.10.024 [10] Lien vers le document http://dx.doi.org/10.1016/j.jhin.2008.10.024 [10]

A case report of Mycoplasma hominis brain abscess identified by MALDI-TOF mass spectrometry.

We report the case of a 43-year-old man with a Mycoplasma hominis brain abscess occurring after a cranial trauma, which was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The presence of colonies on classic blood agar plates and the use of MALDI-TOF MS, a valuable diagnostic tool that identified M. hominis due to its presence in the VITEK MS database, allowed the rapid diagnosis of this infection.